It is recognized that there are many variables that can dictate biological response of tissues that contain radioactivity. Among the many variables are tissue radiosensitivity, distribution of radioactivity at the macroscopic and cellular levels, radiations emitted (e.g. alpha, beta, Auger electrons), and bystander effects. We have a limited understanding of how these variables correlate with biological effects that result from nonuniform distribution of radioactivity. There is mounting evidence that bystander effects play an important role in determining biological response. These are current issues of major importance to human health as it relates to diagnostic and therapeutic nuclear medicine. They have become increasingly urgent to resolve in light of the likelihood of radiological terrorism involving radioactive materials. Over the last several years we have been working toward correlating biological response of tissues containing radioactivity with cellular absorbed dose and variables relating to the bystander effect. We have made substantial progress during our first grant period, including the revelation of important insights into the phenomenology and mechanisms of bystander effects caused by intracellular radioactivity. Our progress will have considerable impact on our capacity to predict the biological effects of incorporated radioactivity. Indeed, our contributions are recognized in the ICRU report on dose specification in nuclear medicine. Our work has also raised important new questions regarding the prediction of response to nonuniform distributions of radioactivity that are addressed in the present proposal. Overall, we hypothesize that the biological response of tissues containing incorporated radionuclides can be correlated with cellular absorbed dose and variables relating to the bystander effect. We will test this hypothesis using a step-wise approach with models of increasing complexity. We will use our original three dimensional (3D) multicellular cluster model to resolve fundamental and significant questions related to the shape of survival dose response curves. Recognizing the limitations of our original model, we have devoted considerable effort toward transitioning our studies on multicellular dosimetry and bystander effects to a new in vitro Cytomatrix model that mimics normal human tissue in vivo. This new 3D model will be used to assess cell cycle alterations, DNA damage, and cell killing caused by nonuniform distributions of radioactivity in both tumor and normal human cell types. Complementing this new model will be development of a theoretical multicellular dosimetry model that blends 3D pCT imaging and stylized analytical models of the cell. This will enable us to test whether our multicellular dosimetry approaches can predict responses in this more complex system. Finally, to initiate transition of our multicellular dosimetry approach to in vivo, we will carry out bystander studies in mouse testis.